Albumin is a major protein of human or animal blood plasma. Clinical use of albumin, as an active ingredient, requires its extraction and purification, which is traditionally carried out by known methods, such as those of Cohn et al. (J. Am. Chem. Soc., 68, 459, 1946) and Kistler et al. (Vox Sang., 7, 1962, 414-424) which are additionally applicable to an industrial scale.
Albumin requirements amount to about 100-300 kg per million inhabitants according to country; for that reason, it is necessary, for clinical purposes, to provide an albumin free from pathogenic viruses and contaminants, which are sources of diseases. Thus, as regards transfusion-transmissible viruses, safety is ensured by viral inactivation methods such as liquid-state pasteurisation of an albumin composition at 60° C. for 10 hours in the presence of a biologically compatible stabiliser (sodium caprylate and/or tryptophanate). This viral safety has been established for more than 60 years and the literature does not report any proven case of virus transmission, e.g. hepatitis B, A or C, or HIV. However, some authors mentioned some cases correlated with the presence, after transfusions, of parvoviruses B 19 in albumin compositions or albumin derivate products. Furthermore, a case has also been reported of an albumin composition that would have led to Creutzfeldt-Jakob disease in a patient having undergone liver transplantation (Créange A., 1995).
Some studies revealed a series of adverse effects occurring after an albumin transfusion due to the presence of lipopolysaccharide substances (pyrogens) in amounts lower than the detection threshold of authorised methods, but capable of occurring during perfusions with large volumes of albumin (e.g. plasma exchanges).
Other side effects are related to the presence of albumin polymers occurring during albumin purification and especially during the above-mentioned pasteurisation step. Others are related to the presence of added stabilisers to prevent the thermal denaturation linked to pasteurisation, especially in patients with allergic backgrounds.
In order to avoid the risk of transmissible infectious agents being present, it has been suggested to produce a so-called “recombinant” albumin, according to U.S. Pat. No. 6,210,683: the gene of albumin is introduced into a host cell, yeast or bacterium, having a high proliferation potential. In turn, this host cell produces albumin in the culture medium or its cytoplasm. This albumin is then separated from the cells by extraction and purified. However, the presence of host cell proteins is often detected and the purification methods must therefore have a very high resolution, which is generally detrimental to the yield. The production cost of a recombinant albumin may then prove to be too high in comparison to that of an albumin generated from plasma.
Another possibility to get round the above-mentioned drawbacks is to implement filtration methods extensively used for the retention of viruses, and chemical or bacteriological contaminants, etc., using qualified filters with variable porosities. One can mention for instance ultrafiltration that has been used from time to time in the absence of other means of virus elimination to ensure the biological safety of an extraction protein such as growth hormone (hGH). The method requires a tangential flow and is only intended for protein solutions or polypeptides having molecular weights equal to or lower than 65 kDa and low concentrations. This leaves chance for viruses to pass through due to the manufacturing imprecision of filtration membranes. Moreover, filter clogging occurs in front operation, resulting in a blockage of the filtrate flow. Filter clogging is all the faster as protein or polypeptide concentration increases.
To increase the viral safety of biological solutions further, it is contemplated to implement nanofiltration, the preferred method for the retention of particles with sizes equal to or greater than about 15-20 nm. This particle retention on qualified filters is easily achieved on a large scale with aqueous solutions or solutions containing small-sized solutes, peptides, amino-acids, mineral ions or organic compounds smaller than about 5 kDa.
The nanofiltration of large-sized solutes has been achieved, for example, with blood coagulation factors, factor IX and factor XI. The article by Burnouf-Radosevich et al. (Vox Sang., 67, p. 132-138, 1994 in reference with Burnouf et al., Vox Sang., 57, p. 225-232, 1989 and Burnouf-Radosevich et al., Transfusion, 22, p. 861-867, 1992) shows that the working conditions of the nanofiltration method imply small volumes of factor IX and factor XI concentrates (3-4 litres), with protein concentrations not exceeding 1 g/L (0.21 g/L and 0.75 g/L, respectively).
Actually, during the nanofiltration process, the large-sized solutes accumulate on the filter, which slows down the filtrate flow until the filter is completely clogged.
The technical solutions to this clogging which consist of implementing a tangential flow nanofiltration, increasing the pressure and changing the directions of the flows do not prove to be efficient because the clogging is progressive and irreversible. An industrial development is therefore greatly compromised because it requires the use of very numerous filters and very large volumes of solutions to be filtered, which generates a prohibitive cost and abnormally increased manufacturing times.
To avoid the clogging phenomenon, a solution consists of adjusting the physicochemical parameters influencing the recovery yield of solutes, while avoiding the passage of contaminants through the filter. Varying these parameters—such as ionic strength, the nature of the solute to be filtered and the pH of the solution to be filtered—as well as the working conditions of the filtration—such as flow rate and pressure—has been the subject of many studies. For instance, the scientific publications by C. Wallis et al., Ann. Rev. Microbial., 33, p. 413-437, 1979 and S. Jacob, Methods of Biochemical Analysis, 22, p. 307-350, 1974, show that the effect of each parameter can individually result in increased or decreased efficiency of virus retention and recovery yield of solutes, and that combining several parameters does not systematically favour a synergy of the effects of improvement of the filtration conditions.